scholarly journals G-Quadruplex Modulation of SP1 Functional Binding Sites at the KIT Proximal Promoter

2020 ◽  
Vol 22 (1) ◽  
pp. 329
Author(s):  
Silvia Da Ros ◽  
Giulia Nicoletto ◽  
Riccardo Rigo ◽  
Silvia Ceschi ◽  
Eleonora Zorzan ◽  
...  
Keyword(s):  
Physiological Mechanism ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Pharmacological Intervention ◽  
Gene Promoters ◽  
Control Of Gene Expression ◽  
G Quadruplex ◽  
Fine Control ◽  
Different Levels

The regulation of conformational arrangements of gene promoters is a physiological mechanism that has been associated with the fine control of gene expression. Indeed, it can drive the time and the location for the selective recruitment of proteins of the transcriptional machinery. Here, we address this issue at the KIT proximal promoter where three G-quadruplex forming sites are present (kit1, kit2 and kit*). On this model, we focused on the interplay between G-quadruplex (G4) formation and SP1 recruitment. By site directed mutagenesis, we prepared a library of plasmids containing mutated sequences of the WT KIT promoter that systematically exploited different G4 formation attitudes and SP1 binding properties. Our transfection data showed that the three different G4 sites of the KIT promoter impact on SP1 binding and protein expression at different levels. Notably, kit2 and kit* structural features represent an on-off system for KIT expression through the recruitment of transcription factors. The use of two G4 binders further helps to address kit2-kit* as a reliable target for pharmacological intervention.

scholarly journals Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter

2004 ◽  
Vol 378 (2) ◽  
pp. 473-484 ◽  
Author(s):  
Stephan RYSER ◽  
Abbas MASSIHA ◽  
Isabelle PIUZ ◽  
Werner SCHLEGEL
Keyword(s):  
Gene Transcription ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Proximal Promoter ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Basal Transcription ◽  
Band Shift ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca2+ responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319–33327].

scholarly journals Ethnic peculiarities of fairy tale folklore of the bulgarians from Moldova as an element of traditional culture in a regional manifestation

2021 ◽  
Author(s):  
Nadejda Cara ◽  
Keyword(s):  
Fairy Tales ◽  
Interdisciplinary Research ◽  
Fairy Tale ◽  
Traditional Culture ◽  
Structural Features ◽  
Ethnic Culture ◽  
Republic Of Moldova ◽  
The Subject ◽  
The Republic ◽  
Different Levels

The article presents some research approaches to the fairy-tale folklore of Bulgarians from the Republic of Moldova. According to the author, the fairy-tales texts of Bulgarians from the Republic of Moldova, their semantic, symbolic, and structural features should be researched as a local (regional) variant of Bulgarian folklore. Identification of ethnocultural markers in the fairy-tales of local Bulgarians on some different levels (such as on the subject, ethno-social and spiritual (church-religious) level) will allow to identify some peculiarities in the adaptation process of Bulgarians that migrated to a new ethnocultural zone, as well as to identify the level of preservation of basic ethnic mentality under the conditions of new “mental environment”. Thus, the study of regional ethnic culture is an interdisciplinary research, which allows discovering how localization in time and space affects ethnic culture, in general, and oral folk art, in particular.

scholarly journals G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders

2020 ◽  
Vol 48 (21) ◽  
pp. 11942-11957
Author(s):  
Giulia Miglietta ◽  
Marco Russo ◽  
Giovanni Capranico
Keyword(s):  
Selective Inhibition ◽  
Telomere Maintenance ◽  
Published Data ◽  
Gene Promoters ◽  
Untranslated Exon ◽  
G Quadruplex ◽  
Functional Consequences ◽  
Hoogsteen Hydrogen Bonds

Abstract Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.

Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level

1991 ◽  
Vol 11 (1) ◽  
pp. 338-343
Author(s):  
D Jefferies ◽  
P Tebabi ◽  
E Pays
Keyword(s):  
Trypanosoma Brucei ◽  
Gene Promoter ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Gene Promoters ◽  
Control Of Gene Expression ◽  
Transient Activity ◽  
Expression Site ◽  
Cat Gene ◽  
Cat Expression

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.

scholarly journals Intra-locked G-quadruplex structures formed by irregular DNA G-rich motifs

2020 ◽  
Vol 48 (6) ◽  
pp. 3315-3327 ◽  
Author(s):  
Arijit Maity ◽  
Fernaldo Richtia Winnerdy ◽  
Weili Denyse Chang ◽  
Gang Chen ◽  
Anh Tuân Phan
Keyword(s):  
Human Genome ◽  
Dna Sequences ◽  
Structural Features ◽  
Nmr Structure ◽  
Solution Nmr ◽  
Structural Studies ◽  
High Propensity ◽  
G Quadruplex ◽  
High Prevalence

Abstract G-rich DNA sequences with tracts of three or more continuous guanines (G≥3) are known to have high propensity to adopt stable G-quadruplex (G4) structures. Bioinformatic analyses suggest high prevalence of G-rich sequences with short G-tracts (G≤2) in the human genome. However, due to limited structural studies, the folding principles of such sequences remain largely unexplored and hence poorly understood. Here, we present the solution NMR structure of a sequence named AT26 consisting of irregularly spaced G2 tracts and two isolated single guanines. The structure is a four-layered G4 featuring two bi-layered blocks, locked between themselves in an unprecedented fashion making it a stable scaffold. In addition to edgewise and propeller-type loops, AT26 also harbors two V-shaped loops: a 2-nt V-shaped loop spanning two G-tetrad layers and a 0-nt V-shaped loop spanning three G-tetrad layers, which are named as VS- and VR-loop respectively, based on their distinct structural features. The intra-lock motif can be a basis for extending the G-tetrad core and a very stable intra-locked G4 can be formed by a sequence with G-tracts of various lengths including several G2 tracts. Findings from this study will aid in understanding the folding of G4 topologies from sequences containing irregularly spaced multiple short G-tracts.

scholarly journals The final steps of [FeFe]-hydrogenase maturation

2019 ◽  
Vol 116 (32) ◽  
pp. 15802-15810 ◽  
Author(s):  
Oliver Lampret ◽  
Julian Esselborn ◽  
Rieke Haas ◽  
Andreas Rutz ◽  
Rosalind L. Booth ◽  
...  
Keyword(s):  
Functional Integration ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Structural Reorganization ◽  
Biologically Inspired ◽  
Hydrogenase Maturation ◽  
Flexible Loop ◽  
Loop Region ◽  
Shed Light ◽  
Secondary Ligand

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.

scholarly journals Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Jung S. Byun ◽  
Samson Park ◽  
Dae Ik Yi ◽  
Jee-Hye Shin ◽  
Sara Gil Hernandez ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Water Solubility ◽  
Binding Protein ◽  
Cancer Cell Line ◽  
Pharmacological Intervention ◽  
Computer Assisted ◽  
Functional Screening ◽  
Gene Promoters ◽  
Breast Cancer Patients ◽  
Qsar Modeling

Abstract The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear proteins that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer-assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro-epithelial genes, preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from gene promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating histone marks at targeted genes. In functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of gene expression in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention.

scholarly journals Initiation of Polyene Macrolide Biosynthesis: Interplay between Polyketide Synthase Domains and Modules as Revealed via Domain Swapping, Mutagenesis, and Heterologous Complementation

2011 ◽  
Vol 77 (19) ◽  
pp. 6982-6990 ◽  
Author(s):  
Sondre Heia ◽  
Sven E. F. Borgos ◽  
Håvard Sletta ◽  
Leticia Escudero ◽  
Elena M. Seco ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Fungal Infections ◽  
Domain Swapping ◽  
Site Directed Mutagenesis ◽  
Active Site Residue ◽  
Polyene Macrolide ◽  
Content Type ◽  
Streptomyces Noursei ◽  
Strict Specificity ◽  
Different Levels

ABSTRACTPolyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT0in the nystatin PKS loading module NysA with the propionate-specific AT1from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT0domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT0and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT0domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of arimA-deficient mutant of the rimocidin/CE-108 producerStreptomyces diastaticus. Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT0domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS ofStreptomyces noursei.

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