scholarly journals Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 277
Author(s):  
Paola Monti ◽  
Vaclav Brazda ◽  
Natália Bohálová ◽  
Otília Porubiaková ◽  
Paola Menichini ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Relative Activity ◽  
Gene Organization ◽  
Functional Assay ◽  
Wild Type ◽  
P53 Family ◽  
Transactivation Potential ◽  
G Quadruplex

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.

scholarly journals Crosstalk between p53 and TGF-β Signalling

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Rebecca Elston ◽  
Gareth J. Inman
Keyword(s):  
Transcriptional Activation ◽  
Cancer Biology ◽  
Target Genes ◽  
Tumour Progression ◽  
Signal Transduction Pathway ◽  
Mirna Biogenesis ◽  
Mutant P53 ◽  
Wild Type ◽  
P53 Family ◽  
Wild Type P53

Wild-type p53 and TGF-β are key tumour suppressors which regulate an array of cellular responses. TGF-β signals in part via the Smad signal transduction pathway. Wild-type p53 and Smads physically interact and coordinately induce transcription of a number of key tumour suppressive genes. Conversely mutant p53 generally subverts tumour suppressive TGF-β responses, diminishing transcriptional activation of key TGF-β target genes. Mutant p53 can also interact with Smads and this enables complex formation with the p53 family member p63 and blocks p63-mediated activation of metastasis suppressing genes to promote tumour progression. p53 and Smad function may also overlap during miRNA biogenesis as they can interact with the same components of the Drosha miRNA processing complex to promote maturation of specific subsets of miRNAs. This paper investigates the crosstalk between p53 and TGF-β signalling and the potential roles this plays in cancer biology.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

scholarly journals DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF.

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996 ◽  
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

scholarly journals A DNA Region Recognized by the Nitric Oxide-Responsive Transcriptional Activator NorR Is Conserved in β- and γ-Proteobacteria

2004 ◽  
Vol 186 (23) ◽  
pp. 7980-7987 ◽  
Author(s):  
Andrea Büsch ◽  
Anne Pohlmann ◽  
Bärbel Friedrich ◽  
Rainer Cramm
Keyword(s):  
Nitric Oxide ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Ralstonia Eutropha ◽  
Upstream Region ◽  
Nitric Oxide Reductase ◽  
Wild Type ◽  
Genome Database ◽  
Nitric Oxide Metabolism ◽  
Signaling Domain

ABSTRACT The σ54-dependent regulator NorR activates transcription of target genes in response to nitric oxide (NO) or NO-generating agents. In Ralstonia eutropha H16, NorR activates transcription of the dicistronic norAB operon that encodes NorA, a protein of unknown function, and NorB, a nitric oxide reductase. A constitutively activating NorR derivative (NorR′), in which the N-terminal signaling domain was replaced by MalE, specifically bound to the norAB upstream region as revealed by gel retardation analysis. Within a 73-bp DNA segment protected by MalE-NorR′ in a DNase I footprint assay, three conserved inverted repeats, GGT-(N7)-ACC (where N is any base), that we consider to be NorR-binding boxes were identified. Mutations altering the spacing or the base sequence of these repeats resulted in an 80 to 90% decrease of transcriptional activation by wild-type NorR. Genome database analyses demonstrate that the GT-(N7)-AC core of the inverted repeat is found in several proteobacteria upstream of gene loci encoding proteins of nitric oxide metabolism, including nitric oxide reductase (NorB), flavorubredoxin (NorV), NO dioxygenase (Hmp), and hybrid cluster protein (Hcp).

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

2005 ◽  
Vol 25 (9) ◽  
pp. 3461-3474 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Fan Zhang ◽  
Gwo Jiunn Hwang ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
General Transcription Factors ◽  
Tata Element ◽  
High Level ◽  
Target Promoters ◽  
Activation Sequence

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

scholarly journals The Influence of Quadruplex Structure in Proximity to P53 Target Sequences on the Transactivation Potential of P53 Alpha Isoforms

2019 ◽  
Vol 21 (1) ◽  
pp. 127
Author(s):  
Otília Porubiaková ◽  
Natália Bohálová ◽  
Alberto Inga ◽  
Natália Vadovičová ◽  
Jan Coufal ◽  
...  
Keyword(s):  
Target Genes ◽  
Tp53 Gene ◽  
Luciferase Reporter ◽  
Biological Processes ◽  
Alternative Promoters ◽  
Quadruplex Dna ◽  
Transactivation Potential ◽  
G Quadruplex ◽  
Damage Response ◽  
Reporter Strains

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.

scholarly journals Differences in Transcriptional Activation by the Two Allelic (L162V Polymorphic) Variants of PPARαafter Omega-3 Fatty Acids Treatment

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-5 ◽  
Author(s):  
Iwona Rudkowska ◽  
Mélanie Verreault ◽  
Olivier Barbier ◽  
Marie-Claude Vohl
Keyword(s):  
Fatty Acids ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Functional Difference ◽  
Omega 3 Fatty Acids ◽  
Omega 3 ◽  
Wild Type ◽  
Allelic Form ◽  
Allelic Variants

Omega-3 fatty acids (FAs) have the potential to regulate gene expression via the peroxisome proliferator-activated receptorα(PPARα); therefore, genetic variations in this gene may impact its transcriptional activity on target genes. It is hypothesized that the transcriptional activity by wild-type L162-PPARαis enhanced to a greater extent than the mutated variant (V162-PPARα) in the presence of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or a mixture of EPA:DHA. To examine the functional difference of the two allelic variants on receptor activity, transient co-transfections were performed in human hepatoma HepG2 cells activated with EPA, DHA and EPA:DHA mixtures. Results indicate that the addition of EPA or DHA demonstrate potential to increase the transcriptional activity by PPARαwith respect to basal level in both variants. Yet, the EPA:DHA mixtures enhanced the transcriptional activity to a greater extent than individual FAs indicating possible additive effects of EPA and DHA. Additionally, the V162 allelic form of PPARαdemonstrated consistently lower transcriptional activation when incubated with EPA, DHA or EPA:DHA mixtures than, the wild-type variant. In conclusion, both allelic variants of the PPARαL162V are activated by omega-3 FAs; however, the V162 allelic form displays a lower transcriptional activity than the wild-type variant.

scholarly journals Transcriptional activation of the wild-type and mutant vitamin D receptors by vitamin D3 analogs

2016 ◽  
Vol 57 (1) ◽  
pp. 23-32
Author(s):  
Kumi Futawaka ◽  
Tetsuya Tagami ◽  
Yuki Fukuda ◽  
Rie Koyama ◽  
Ayaka Nushida ◽  
...  
Keyword(s):  
Vitamin D ◽  
Vitamin D3 ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Active Form ◽  
Therapeutic Effects ◽  
Wild Type ◽  
Human Embryonic Kidney Cells ◽  
Vitamin D Receptors ◽  
High Doses

The active form of vitamin D3 (1α,25(OH)2D3, also known as calcitriol) controls the expression of target genes via the vitamin D receptor (VDR). Vitamin D-dependent rickets type II (VDDRII) is a congenital disease caused by inactivating mutations in the VDR. The condition is treated with high doses of calcitriol, but the therapeutic effects of other synthetic VD3 analogs have not yet been investigated. In the present study, we analyzed the transcriptional activity of seven different VD3 analogs with VDRs carrying ligand-binding domain mutations identified in VDDRII patients. Wild-type VDR (WT-VDR) and seven mutant VDRs were expressed in TSA201 human embryonic kidney cells, HepG2 human liver cancer cells, and MC3T3-E1 mouse calvaria cells, and their transcriptional activation with VD3 analogs were analyzed by performing transient expression assays, western blotting, and quantitative real-time PCR. The results demonstrated that falecalcitriol stimulated significantly higher transcriptional activation of the WT-VDR and some mutant VDRs than did calcitriol. Calcitriol showed almost no transcriptional activation of the VDR with the I268T mutation identified in a severe case of VDDRII, whereas falecalcitriol caused a dose-dependent increase in the activation of this mutant VDR. Our findings demonstrate that falecalcitriol has a VDR activation profile distinct from that of calcitriol and may exhibit therapeutic effects even on difficult-to-treat VDDRII cases resistant to calcitriol. It is also possible that VDDRII patients responding to high doses of calcitriol could be appropriately treated with low doses of falecalcitriol.

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