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.

scholarly journals LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Run Jin ◽  
Samantha Klasfeld ◽  
Yang Zhu ◽  
Meilin Fernandez Garcia ◽  
Jun Xiao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Fate ◽  
Chromatin Accessibility ◽  
Binding Motifs ◽  
Chromatin Remodelers ◽  
Pioneer Transcription Factor ◽  
Chromatin Reprogramming

AbstractMaster transcription factors reprogram cell fate in multicellular eukaryotes. Pioneer transcription factors have prominent roles in this process because of their ability to contact their cognate binding motifs in closed chromatin. Reprogramming is pervasive in plants, whose development is plastic and tuned by the environment, yet little is known about pioneer transcription factors in this kingdom. Here, we show that the master transcription factor LEAFY (LFY), which promotes floral fate through upregulation of the floral commitment factor APETALA1 (AP1), is a pioneer transcription factor. In vitro, LFY binds to the endogenous AP1 target locus DNA assembled into a nucleosome. In vivo, LFY associates with nucleosome occupied binding sites at the majority of its target loci, including AP1. Upon binding, LFY ‘unlocks’ chromatin locally by displacing the H1 linker histone and by recruiting SWI/SNF chromatin remodelers, but broad changes in chromatin accessibility occur later. Our study provides a mechanistic framework for patterning of inflorescence architecture and uncovers striking similarities between LFY and animal pioneer transcription factor.

scholarly journals LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate

2020 ◽  
Author(s):  
Run Jin ◽  
Samantha Klasfeld ◽  
Meilin Fernandez Garcia ◽  
Jun Xiao ◽  
Soon-Ki Han ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Fate ◽  
Chromatin Accessibility ◽  
Binding Motifs ◽  
Chromatin Remodelers ◽  
Pioneer Transcription Factor ◽  
Chromatin Reprogramming

ABSTRACTMaster transcription factors reprogram cell fate in multicellular eukaryotes. Pioneer transcription factors have prominent roles in this process because of their ability to contact their cognate binding motifs in closed chromatin. Reprogramming is pervasive in plants, whose development is plastic and tuned by the environment, yet no bonafide pioneer transcription factor has - been identified in this kingdom. Here we show that the master transcription factor LEAFY (LFY), which promotes floral fate through upregulation of the floral commitment factor APETALA1 (AP1), is a pioneer transcription factor. In vitro, LFY binds in a sequence-specific manner and with high affinity to the endogenous AP1 target locus DNA assembled into a nucleosome. In vivo, LFY associates with nucleosome occupied binding sites at the majority of its target loci, including AP1, where it co-occupies DNA with histones. Moreover, the LFY DNA contact helix shares defining properties with those of strong nucleosome binding pioneer factors. At the AP1 locus, LFY unlocks chromatin locally by displacing the H1 linker histone and by recruiting SWI/SNF chromatin remodelers, but broad changes in chromatin accessibility occur later and require activity of additional, non-pioneer transcription, factors. Our study provides a mechanistic framework for patterning of inflorescence architecture and uncovers striking similarities between plant and animal pioneer transcription factors. Further analyses aimed at elucidating the defining characteristics of pioneer transcription factors will allow harnessing these for enhanced cell fate reprogramming.

scholarly journals The molecular logic of Nanog-induced self-renewal

2018 ◽  
Author(s):  
Victor Heurtier ◽  
Nick Owens ◽  
Inma Gonzalez ◽  
Florian Mueller ◽  
Caroline Proux ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Self Renewal ◽  
Pluripotency Factors ◽  
Molecular Logic ◽  
Repressive Histone Mark ◽  
Gene Regulatory

Transcription factor networks, together with histone modifications and signalling pathways, underlie the establishment and maintenance of gene regulatory architectures associated with the molecular identity of each cell type. However, how master transcription factors individually impact the epigenomic landscape and orchestrate the behaviour of regulatory networks under different environmental constraints is only very partially understood. Here, we show that the transcription factor Nanog deploys multiple distinct mechanisms to enhance embryonic stem cell self-renewal. In the presence of LIF, which fosters self-renewal, Nanog rewires the pluripotency network by promoting chromatin accessibility and binding of other pluripotency factors to thousands of enhancers. In the absence of LIF, Nanog blocks differentiation by sustaining H3K27me3, a repressive histone mark, at developmental regulators. Among those, we show that the repression of Otx2 plays a preponderant role. Our results underscore the versatility of master transcription factors, such as Nanog, to globally influence gene regulation during developmental processes.

scholarly journals The future of toxicity testing: a focus on in vitro methods using a quantitative high-throughput screening platform

2010 ◽  
Vol 15 (23-24) ◽  
pp. 997-1007 ◽  
Author(s):  
Sunita J. Shukla ◽  
Ruili Huang ◽  
Christopher P. Austin ◽  
Menghang Xia
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Toxicity Testing ◽  
In Vitro Methods ◽  
The Future ◽  
Screening Platform

scholarly journals Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors

2018 ◽  
Vol 24 (1) ◽  
pp. 77-85
Author(s):  
Simon L. Currie ◽  
Steven L. Warner ◽  
Hariprasad Vankayalapati ◽  
Xiaohui Liu ◽  
Sunil Sharma ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Transcription Factors ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Assays ◽  
Ets Transcription Factors ◽  
Virtual Screen ◽  
Prostate Cells ◽  
Screening Assays

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

Recent Advances in High-throughput Platforms with Engineered Biomaterial Microarrays for Screening of Cell and Tissue Behavior

2019 ◽  
Vol 24 (45) ◽  
pp. 5458-5470 ◽  
Author(s):  
Kijun Park ◽  
Yeontaek Lee ◽  
Jungmok Seo
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Continuous Monitoring ◽  
In Vitro Screening ◽  
Cellular Behavior ◽  
Current Trends ◽  
Recent Advances ◽  
Screening Platform ◽  
Review Current

In the last decades, bioengineers have developed myriad biomaterials for regenerative medicine. Development of screening techniques is essential for understanding complex behavior of cells in the biological microenvironments. Conventional approaches to the screening of cellular behavior in vitro have limitations in terms of accuracy, reusability, labor-intensive screening, and versatility. Thus, drug screening and toxicology test through in vitro screening platforms have been underwhelming. Recent advances in the high-throughput screening platforms somewhat overcome the limitations of in vitro screening platforms via repopulating human tissues’ biophysical and biomchemical microenvironments with the ability to continuous monitoring of miniaturized human tissue behavior. Herein, we review current trends in the screening platform in which a high-throughput system composed of engineered microarray devices is developed to investigate cell-biomaterial interaction. Furthermore, diverse methods to achieve continuous monitoring of cell behavior via developments of biosensor integrated high-throughput platforms, and future perspectives on high-throughput screening will be provided.

scholarly journals Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors

2017 ◽  
Author(s):  
Simon L. Currie ◽  
Stephen L. Warner ◽  
Hariprasad Vankayalapati ◽  
Xiao-Hui Liu ◽  
Sunil Sharma ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Transcription Factors ◽  
High Throughput ◽  
High Throughput Screening ◽  
Ets Transcription Factors ◽  
Lower Affinity ◽  
Virtual Screen ◽  
Prostate Cells ◽  
Screening Assays

AbstractETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we develop two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable for high throughput screening. Using ETS1 as a model, these assays were applied to screen 110 compounds derived from a high-throughput virtual screen. We find that the use of lower affinity DNA-binding sequences, similar to those which ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

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