Transcription Factor-Based Biosensors in High-Throughput Screening: Advances and Applications

2018 ◽  
Vol 13 (7) ◽  
pp. 1700648 ◽  
Author(s):  
Feng Cheng ◽  
Xiao-Ling Tang ◽  
Tsvetan Kardashliev
Keyword(s):  
Transcription Factor ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening

2011 ◽  
Vol 103 (12) ◽  
pp. 962-978 ◽  
Author(s):  
Patrick J. Grohar ◽  
Girma M. Woldemichael ◽  
Laurie B. Griffin ◽  
Arnulfo Mendoza ◽  
Qing-Rong Chen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Oncogenic Transcription Factor

scholarly journals Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

2019 ◽  
Author(s):  
Christina K. Kim ◽  
Kelvin F. Cho ◽  
Min Woo Kim ◽  
Alice Y. Ting
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Protein ◽  
Protein Protein Interactions ◽  
Lov Domain ◽  
Omics Technologies ◽  
Lower Background

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested “AND” gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

scholarly journals Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Christina K Kim ◽  
Kelvin F Cho ◽  
Min Woo Kim ◽  
Alice Y Ting
Keyword(s):  
Transcription Factor ◽  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Cellular Protein ◽  
Protein Protein Interactions ◽  
Lov Domain ◽  
Omics Technologies ◽  
Lower Background

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested ‘AND’ gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.

Chemical Genomic Screen Identifies Ionophores as Modulators of Notch1 in T-ALL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 200-200
Author(s):  
Giovanni Roti ◽  
Kenneth N. Ross ◽  
Cristina Mecucci ◽  
Adolfo A. Ferrando ◽  
Jon C. Aster ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Ion Channel ◽  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Screening ◽  
Detection System ◽  
Lymphoblastic Leukemia ◽  
Proteolytic Activation ◽  
Ion Channel Modulators

Abstract Although in recent years intensive chemotherapy regimens have improved outcome for patients with T-cell acute lymphoblastic leukemia (T-ALL), side effects and relapse-rates remain high. An alternative therapeutic approach would be to target constitutively active mutated Notch1. Notch1, a trans-membrane receptor that acts as a transcription factor following proteolytic activation by the γ-secretase complex, harbors gain of function mutations in over 50% of T-ALL. Early clinical trials attempting to modulate Notch1 with γ-secretase inhibitors (GSI) were closed secondary to gastrointestinal toxicity, suggesting that alternative approaches to inhibiting Notch1 signaling are needed. Transcription factor complexes are difficult to target pharmacologically with conventional high-throughput screening approaches. We therefore sought to target Notch1 with a new gene expressionbased approach to small molecule library screening. Signatures for the Notch1 on versus off states were defined using microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a GSI (Notch1 off). We adapted this 32-gene signature to our gene expression-based high-throughput screening (GE-HTS) assay, which uses ligation-mediated amplification (LMA) and a Luminex bead-based detection system. As proof of principle, we confirmed that an shRNA against Notch1 induced the Notch1 off signature in DND41 cells (a T-ALL cell line bearing a mutated Notch1 allele). In order to facilitate future clinical translation, we screened a collection of 4,500 bioactive compounds highly enriched for FDA-approved drugs in DND41 cells. Twenty-eight compounds that induced the Notch1 off signature were identified as top hits. These compounds were tested in a secondary screen in a 2-fold dilution series in two GSI sensitive cell lines, DND41 and KOPTK1, and one GSI resistant cell line, PF382. Ion channel modulators emerged as the top class of compounds confirmed to induce the Notch1 off signature across all cell lines. We next focused on two representative compounds, the ionophores salinomycin and ionomycin. Similar to GSI, both of these molecules induced cell cycle arrest and markedly decreased levels of the activated intracellular portion of Notch (ICN). Both salinomycin and ionomycin, as well as numerous other ionophores that scored as inhibitors in the screen, are known to increase cytosolic calcium. These studies are consistent with a role for calcium in the activation of oncogenic forms of Notch1 and suggest a potential role for ion channel modulators in the treatment of T-ALL.

scholarly journals Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast

2020 ◽  
Author(s):  
Emil D. Jensen ◽  
Francesca Ambri ◽  
Marie B. Bendtsen ◽  
Alex A. Javanpour ◽  
Chang C. Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Biosynthetic Pathway ◽  
A Priori ◽  
Enzymatic Reaction ◽  
Search Space ◽  
Muconic Acid ◽  
Metabolic Reactions

SummaryDirected evolution is a powerful method to optimize proteins and metabolic reactions towards user-defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection, and amplification. While powerful, systematic searches through large sequence-spaces is a labor-intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generates enzyme variants using the recently developed orthogonal replication system, OrthoRep, and screens for optimal performance in high-throughput using a transcription factor-based biosensor. We demonstrate the strengths of this workflow by evolving a ratelimiting enzymatic reaction of the biosynthetic pathway for cis,cis-muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high-throughput sorting of best-performing variants using a transcription factor-based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers >13-fold compared to reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study, is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.

scholarly journals A high-throughput genome-integrated assay reveals spatial dependencies governing Tcf7l2 binding

2020 ◽  
Author(s):  
Tomasz Szczesnik ◽  
Lendy Chu ◽  
Joshua W. K. Ho ◽  
Richard Sherwood
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
High Throughput ◽  
Dna Sequences ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Genomic Locus ◽  
Screening Platform

2 SummaryPredicting where transcription factors bind in the genome from their in-vitro DNA binding affinity is confounded by the large number of possible interactions with nearby transcription factors. To characterise the binding logic for the Wnt effector transcription factor Tcf7l2, we have developed a high-throughput screening platform in which thousands of 99-bp synthesised DNA sequences are inserted into a specific genomic locus through CRISPR/Cas9-based homology-directed repair, followed by measurement of Tcf7l2 binding by DamID. Using this platform at two genomic loci in mouse embryonic stem cells, we show that while the binding of Tcf7l2 closely follows the in-vitro motif binding strength and is influenced by local chromatin accessibility, it is also strongly affected by the surrounding 99-bp of sequence. The presence of nearby Oct4 and Klf4 motifs promote Tcf7l2 binding, particularly in the adjacent ~20 to 50-bp nearby and oscillating with a 10.8-bp phasing relative to these cofactor motifs, which matches the turn of a DNA helix. This novel high-throughput DamID assay provides a powerful platform to determine local DNA sequence grammars that causally influence transcription factor binding in controlled genomic contexts.

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