scholarly journals TBP and SNAP50 transcription factors bind specifically to the Pr77 promoter sequence from trypanosomatid non-LTR retrotransposons

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Francisco Macías ◽  
Raquel Afonso-Lehmann ◽  
Patricia E. Carreira ◽  
M. Carmen Thomas
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Direct Interaction ◽  
Promoter Sequence ◽  
Nuclear Proteins ◽  
Hill Coefficient ◽  
Small Nuclear Rna ◽  
Mobile Dna ◽  
Mobility Shift

Abstract Background Trypanosomatid genomes are colonized by active and inactive mobile DNA elements, such as LINE, SINE-like, SIDER and DIRE retrotransposons. These elements all share a 77-nucleotide-long sequence at their 5′ ends, known as Pr77, which activates transcription, thereby generating abundant unspliced and translatable transcripts. However, transcription factors that mediates this process have still not been reported. Methods TATA-binding protein (TBP) and small nuclear RNA-activating protein 50 kDa (SNAP50) recombinant proteins and specific antibodies raised against them were generated. Protein capture assay, electrophoretic mobility-shift assays (EMSA) and EMSA competition assays carried out using these proteins and nuclear proteins of the parasite together to specific DNA sequences used as probes allowed detecting direct interaction of these transcription factors to Pr77 sequence. Results This study identified TBP and SNAP50 as part of the DNA-protein complex formed by the Pr77 promoter sequence and nuclear proteins of Trypanosoma cruzi. TBP establishes direct and specific contact with the Pr77 sequence, where the DPE and DPE downstream regions are docking sites with preferential binding. TBP binds cooperatively (Hill coefficient = 1.67) to Pr77 and to both strands of the Pr77 sequence, while the conformation of this highly structured sequence is not involved in TBP binding. Direct binding of SNAP50 to the Pr77 sequence is weak and may be mediated by protein–protein interactions through other trypanosomatid nuclear proteins. Conclusions Identification of the transcription factors that mediate Pr77 transcription may help to elucidate how these retrotransposons are mobilized within the trypanosomatid genomes and their roles in gene regulation processes in this human parasite. Graphic abstract

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

scholarly journals Inhibitory Effects of Bangladeshi Medicinal Plant Extracts on Interactions between Transcription Factors and Target DNA Sequences

2008 ◽  
Vol 5 (3) ◽  
pp. 303-312 ◽  
Author(s):  
Ilaria Lampronti ◽  
Mahmud T. H. Khan ◽  
Monica Borgatti ◽  
Nicoletta Bianchi ◽  
Roberto Gambari
Keyword(s):  
Transcription Factors ◽  
Medicinal Plants ◽  
Cell Lines ◽  
Plant Extracts ◽  
Dna Sequences ◽  
Cell Cycle Progression ◽  
Biological Effects ◽  
Mobility Shift ◽  
Medicinal Plant Extracts ◽  
Target Dna

Several transcription factors (TFs) play crucial roles in governing the expression of different genes involved in the immune response, embryo or cell lineage development, cell apoptosis, cell cycle progression, oncogenesis, repair and fibrosis processes and inflammation. As far as inflammation, TFs playing pivotal roles are nuclear factor kappa B (NF-kB), activator protein (AP-1), signal transducer and activator of transcription (STATs), cAMP response element binding protein (CREB) and GATA-1 factors. All these TFs regulate the expression of pro-inflammatory cytokines and are involved in the pathogenesis of a number of human disorders, particularly those with an inflammatory component. Since several medicinal plants can be employed to produce extracts exhibiting biological effects and because alteration of gene transcription represents a very interesting approach to control the expression of selected genes, this study sought to verify the ability of several extracts derived from Bangladeshi medicinal plants in interfering with molecular interactions between different TFs and specific DNA sequences. We first analyzed the antiproliferative activity of 19 medicinal plants on different human cell lines, including erythroleukemia K562, B lymphoid Raji and T lymphoid Jurkat cell lines. Secondly, we employed the electrophoretic mobility shift assay as a suitable technique for a fast screening of plant extracts altering the binding between NF-kB, AP-1, GATA-1, STAT-3, CREB and the relative target DNA elements.

scholarly journals Cloning of Interferon-Stimulated Gene 17: The Promoter and Nuclear Proteins That Regulate Transcription

1999 ◽  
Vol 13 (7) ◽  
pp. 1197-1206 ◽  
Author(s):  
David J. Perry ◽  
Kathy J. Austin ◽  
Thomas R. Hansen
Keyword(s):  
Transcription Factors ◽  
Electrophoretic Mobility Shift Assay ◽  
Early Pregnancy ◽  
General Structure ◽  
Nuclear Proteins ◽  
Signal Transducer ◽  
Regulatory Factor ◽  
Mobility Shift ◽  
Uterine Endometrium ◽  
Mobility Shift Assay

Abstract A member of the interferon-stimulated gene (ISG) family encodes a 17-kDa ubiquitin homolog called ISG17 that is induced in the bovine uterine endometrium by interferon-τ (IFN-τ) during early pregnancy. The bovine (b) ISG17 cDNA shares 30% identity with a tandem ubiquitin repeat and 70% identity with human (h) ISG15. The present experiments were designed to sequence the bISG17 gene, compare general structure with the hISG15 gene, and to identify transcription factors that were induced by IFN-τ in bovine endometrial (BEND) cells. The promoter of the bISG17 gene was similar to the hISG15 gene in placement of a tandem IFN-stimulatory response element (ISRE) at position −90, but unique in the presence of three additional ISREs at positions −123, −332, and −525. IFN-τ (25 nm) induced nuclear proteins in BEND cells that interacted with a tandem bISG17 ISRE in electrophoretic mobility shift assay (EMSA). IFN-regulatory factor-1 (IRF-1) bound to this ISRE based upon supershift EMSA using antiserum against IRF-1. IFN-τ activated STAT-1 (signal transducer and activator of transcription-1) and -2 by 0.5 h, and IRF-1 by 2 h in BEND cells. It is concluded that the bISG17 gene is similar to the hISG15 gene, retains an ISRE that interacts with IRF-1, and is possibly induced initially by the STATs and later by IRF-1 in response to IFN-τ during early pregnancy.

scholarly journals A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC

2012 ◽  
Vol 194 (23) ◽  
pp. 6431-6440 ◽  
Author(s):  
Rodrigo Sieira ◽  
Gastón M. Arocena ◽  
Angeles Zorreguieta ◽  
Diego J. Comerci ◽  
Rodolfo A. Ugalde
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Cell Envelope ◽  
Regulatory Element ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Mobility Shift ◽  
Content Type ◽  
Environmental Signals

ABSTRACTType IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genusBrucellaessentially depends on the integrity of the T4SS-encodingvirBoperon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of thevirBgenes that were isolated from total protein extracts ofBrucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of thevirBpromoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNAin vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activatedvirBexpression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of thevirBpromoter and is probably involved in coordinating gene expression in response to specific environmental signals.

Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

scholarly journals Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter

2009 ◽  
Vol 418 (1) ◽  
pp. 103-112 ◽  
Author(s):  
Marianne A. Grant ◽  
Rebecca M. Baron ◽  
Alvaro A. Macias ◽  
Matthew D. Layne ◽  
Mark A. Perrella ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
High Mobility ◽  
Promoter Sequence ◽  
Therapeutic Benefit ◽  
Mobility Shift ◽  
Beneficial Effects ◽  
The Core

The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with Gram-negative bacteria that elaborate endotoxin. The HMGA1 (high-mobility group A1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct (octamer) sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule MGB (minor-groove binder) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. We therefore hypothesized that netropsin would improve survival from murine endotoxaemia by attenuating NOS2 induction through interference with HMGA1 DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxaemia in wild-type mice, yet not in NOS2-deficient mice, supporting an important role for NOS2 in the beneficial effects of MGB administration. Netropsin significantly attenuated NOS2 promoter activity in macrophage transient transfection studies and the AT-rich HMGA1 DNA-binding site was critical for this effect. EMSAs (electrophoretic mobility-shift assays) demonstrated that netropsin interferes with HMGA1 NOS2 promoter binding and NMR spectroscopy was undertaken to characterize this disruption. Chemical shift perturbation analysis identified that netropsin effectively competes both HMGA1 DNA-binding AT-hooks from the AT-rich NOS2 promoter sequence. Furthermore, NOESY data identified direct molecular interactions between netropsin and A/T base pairs within the NOS2 promoter HMGA1-binding site. Finally, we determined a structure of the netropsin/NOS2 promoter Oct site complex from molecular modelling and dynamics calculations. These findings represent important steps toward refined structure-based ligand design of novel compounds for therapeutic benefit that can selectively target key regulatory regions within genes that are important for the development of critical illness.

scholarly journals Dual Roles for Pax-6: a Transcriptional Repressor of Lens Fiber Cell-Specific β-Crystallin Genes

1998 ◽  
Vol 18 (9) ◽  
pp. 5579-5586 ◽  
Author(s):  
Melinda K. Duncan ◽  
John I. Haynes ◽  
Ales Cvekl ◽  
Joram Piatigorsky
Keyword(s):  
Epithelial Cells ◽  
Promoter Activity ◽  
Direct Interaction ◽  
Promoter Sequence ◽  
Full Length ◽  
Lens Epithelial Cells ◽  
Lens Fiber ◽  
High Concentration ◽  
Mobility Shift ◽  
Lens Fiber Cell

ABSTRACT It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the αB-, αA-, δ1-, and ζ-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and β-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the βB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed βB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (−183 to −152, −120 to −48, and −30 to +1) of the βB1-crystallin promoter. Earlier experiments showed that the βB1-crystallin promoter sequence from −120 to −48 contains a cis element (PL2 at −90 to −76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the βB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of βB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.

Differential Binding of Creb, Usf, and c-myc to the Calreticulin Human Specific –220c May Be Linked with the Evolution of Higher Brain Functions in Human

2016 ◽  
Vol 33 (S1) ◽  
pp. S465-S466
Author(s):  
S. Farashi
Keyword(s):  
Transcription Factors ◽  
Protein Complexes ◽  
Promoter Sequence ◽  
Mobility Shift ◽  
Brain Functions ◽  
Differential Binding ◽  
Mobility Shift Assay ◽  
Competing Interest ◽  
Higher Brain Functions ◽  
Human Specific

IntroductionWe have previously reported a human-specific nucleotide in the promoter sequence of the calreticulin (CALR) gene at position –220C, which is the site of action of valproic acid.ObjectivesReversion of this nucleotide to the ancestral type, –220A, co-occurs with severe deficit in higher brain cognitive functions.AimsIn the current study, we compare the pattern of protein binding between –220C and –220A.MethodsAntibodies reactive against transcription factors CREB, USF, and c-Myc were used to identify the specific proteins involved in complexes with DNA using electrophoretic mobility shift assay (EMSA).ResultsSignificant increase was observed in the overall protein complexes binding to the –220 C allele vs. –220A. The transcription factors, CREB, USF, and c-Myc, were differentially bound to –220C, represented by supershifts.ConclusionsWe propose that differential binding of CREB, USF, and c-Myc to CALR nucleotide –220C may be linked with the evolution of higher brain functions in human.Disclosure of interestThe author has not supplied his/her declaration of competing interest.

scholarly journals Bending DNA can repress a eukaryotic basal promoter and inhibit TFIID binding.

1995 ◽  
Vol 15 (10) ◽  
pp. 5492-5498 ◽  
Author(s):  
A TenHarmsel ◽  
M D Biggin
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Promoter Sequence ◽  
Eukaryotic Transcription ◽  
Transcription System ◽  
General Transcription Factors ◽  
Dna Loop ◽  
Reduced Rate

Previous studies indicated that repression by eve involves cooperative DNA binding and leads to the formation of a DNA loop which encompasses the DNA sequences normally bound by the RNA polymerase II general transcription factors. To test the general principle of whether bending of a basal promoter sequence can contribute directly to repression of transcription, a minicircle template of 245 bp was used. In a purified transcription system, transcription from the minicircular DNA is greatly reduced compared with that from the identical DNA fragment in linear form. Transcription is also reduced when the minicircle contains a single-stranded nick, indicating that transcription is reduced because of DNA bending, rather than any constraint on supercoiling. We show that the reduced transcription from the minicircle in these experiments is not due to a reduced rate of elongation by RNA polymerase II. Rather, repression occurs, at least in part, because binding of the general transcription factor TFIID to the minicircle is strongly inhibited compared with binding to the linear DNA. We suggest that bending DNA may be a mechanism by which eukaryotic transcription may be regulated, by modulating the activity of the general transcription factors.

scholarly journals Plant Transcription Factors Involved in Drought and Associated Stresses

2021 ◽  
Vol 22 (11) ◽  
pp. 5662
Author(s):  
Maria Hrmova ◽  
Syed Sarfraz Hussain
Keyword(s):  
Transcription Factors ◽  
Stress Tolerance ◽  
Protein Interactions ◽  
Dna Sequences ◽  
Leucine Zipper ◽  
Plant Stress ◽  
3D Structure ◽  
Post Translational Modification ◽  
Basic Leucine Zipper ◽  
The Impact

Transcription factors (TFs) play a significant role in signal transduction networks spanning the perception of a stress signal and the expression of corresponding stress-responsive genes. TFs are multi-functional proteins that may simultaneously control numerous pathways during stresses in plants—this makes them powerful tools for the manipulation of regulatory and stress-responsive pathways. In recent years, the structure-function relationships of numerous plant TFs involved in drought and associated stresses have been defined, which prompted devising practical strategies for engineering plants with enhanced stress tolerance. Vast data have emerged on purposely basic leucine zipper (bZIP), WRKY, homeodomain-leucine zipper (HD-Zip), myeloblastoma (MYB), drought-response elements binding proteins/C-repeat binding factor (DREB/CBF), shine (SHN), and wax production-like (WXPL) TFs that reflect the understanding of their 3D structure and how the structure relates to function. Consequently, this information is useful in the tailored design of variant TFs that enhances our understanding of their functional states, such as oligomerization, post-translational modification patterns, protein-protein interactions, and their abilities to recognize downstream target DNA sequences. Here, we report on the progress of TFs based on their interaction pathway participation in stress-responsive networks, and pinpoint strategies and applications for crops and the impact of these strategies for improving plant stress tolerance.

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