A yeast one‐hybrid and microfluidics‐based pipeline to map mammalian gene regulatory networks

Gubelmann, Carine; Waszak, Sebastian M.; Isakova, Alina; Holcombe, Wiebke; Hens, Korneel; Iagovitina, Antonina; Feuz, Jean-Daniel; Raghav, Sunil K.; Simicevic, Jovan; Deplancke, Bart

doi:10.1038/msb.2013.38

Cited by 35 publications

(33 citation statements)

References 90 publications

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“…A pENTR library containing the coding sequences of 750 mouse transcription factors (TFs) without STOP codon (Gubelmann et al, 2013) was recombined in the doxycycline-inducible expression vector described in (Deluz et al, 2016), (hereafter referred as to pLVTRE3G-GW-HA/YPet). This vector allows for dox-inducible expression of each TF fused either to a YPet (a yellow fluorescent protein) or to three HA tags, depending on the presence or absence of Cre recombinase, respectively.…”

Section: Dna Constructsmentioning

confidence: 99%

Mitotic chromosome binding predicts transcription factor properties in interphase

Raccaud

Alber

Friman

et al. 2018

Preprint

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Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequencespecific/non-specific DNA binding affinity. How these properties affect the ability of TFs to occupy their specific binding sites in the genome and modify the epigenetic landscape is unclear. Here we combined live cell quantitative measurements of mitotic chromosome binding (MCB) of 502 TFs, measurements of TF mobility by fluorescence recovery after photobleaching, single molecule imaging of DNA binding in live cells, and genome-wide mapping of TF binding and chromatin accessibility. MCB scaled with interphase properties such as association with DNA-rich compartments, mobility, as well as large differences in genome-wide specific site occupancy that correlated with TF impact on chromatin accessibility. As MCB is largely mediated by electrostatic, non-specific TF-DNA interactions, our data suggests that non-specific DNA binding of TFs enhances their search for specific sites and thereby their impact on the accessible chromatin landscape.

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Section: Dna Constructsmentioning

confidence: 99%

Mitotic chromosome binding predicts transcription factor properties in interphase

Raccaud

Alber

Friman

et al. 2018

Preprint

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“…Yeast one-hybrid (Y1H) is an in vivo method complementary to ChIP and DHS analyses in the sense that it allows for the discovery of which TFs are able to bind to a DNA sequence of interest (gene-centred approach), instead of the sequences bound by a TF of interest (TF-centred approach) [41]. The technique also provides a good starting point to study transcriptional regulatory circuits in cells as described in both animals [42,43] and plants [44]. Despite a number of recently developed techniques to study TF-DNA interaction, one of the most important challenges remains: how can we achieve sufficient spatial and temporal resolutions to capture the TF binding event in cells?…”

Section: In Vitro Assays Provide a Molecular Understanding Of How Tfsmentioning

confidence: 99%

“Hit‐and‐run”: Transcription factors get caught in the act

2015

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A key challenge for understanding transcriptional regulation is being able to measure transcription factor (TF)-DNA binding events with sufficient spatial and temporal resolution; that is, when and where TFs occupy their cognate sites. A recent study by Para et al. has highlighted the dynamics underlying the activation of gene expression by a master regulator TF. This study provides concrete evidence for a long-standing hypothesis in biology, the "hit-and-run" mechanism, which was first proposed decades ago. That is, gene expression is dynamically controlled by a TF that transiently binds and activates a target gene, which might stay in a transcriptionally active state after the initial binding event has ended. Importantly, the experimental procedure introduced, TARGET, provides a useful way for identifying multiple target genes transiently bound by their regulators, which can be used in conjunction with other well-established methods to improve our understanding of transcriptional regulatory dynamics.

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“…Recent availability of genome sequences enabled the construction of genome‐wide collections of plasmids carrying gene open reading frames (ORFeomes) (Yamada et al., ). ORFeome collections of TF‐AD encoding plasmids, which include most TFs encoded in a genome (Burdo et al., ; Fuxman Bass et al., ; Gaudinier et al., ; Gubelmann et al., ; Hens et al., ; King, Foster, Davison, Rawls, & Breton, ; Pruneda‐Paz et al., ; Reboul et al., ; Reece‐Hoyes et al., ), were instrumental to develop improved Y1H strategies called gene‐centered Y1H assays (Deplancke et al., ; Deplancke, Dupuy, Vidal, & Walhout, ; Pruneda‐Paz et al., ; Pruneda‐Paz, Breton, Para, & Kay, ; Reece‐Hoyes et al., ; Vermeirssen et al., ). In the approach that has been most widely used, individual clones in a TF‐AD ORFeome collection are organized in an arrayed multi‐well format.…”

Section: Introductionmentioning

confidence: 99%

“…High‐throughput approaches were developed to perform the aforementioned gene‐centered Y1H (HT‐Y1H) assay using large TF ORFeome collections and to facilitate the simultaneous survey of multiple promoter baits (Gubelmann et al., ; Hens, Feuz, & Deplancke, ; Kang, Breton, & Pruneda‐Paz, ; Pruneda‐Paz et al., ; Reece‐Hoyes et al., ; Vermeirssen et al., ). Many of these approaches rely on automation compatible protocols, either in solid or liquid multi‐well arrayed formats, to deliver a TF ORFeome clone collection into reporter strains and to analyze the reporter gene activity in the resulting cells (Kang et al., ; Pruneda‐Paz et al., ; Reece‐Hoyes et al., ).…”

Section: Introductionmentioning

confidence: 99%

High‐Throughput Yeast One‐Hybrid Screens Using a Cell Surface gLUC Reporter

Bonaldi

Kang

et al. 2019

CP Plant Biology

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Gene‐centered yeast one‐hybrid (Y1H) screens using arrayed genome‐wide transcription factor (TF) clone collections provide a simple and effective strategy to identify TF‐promoter interactions using a DNA fragment as bait. In an effort to improve the assay we recently developed a Y1H system that uses a cell surface Gaussia luciferase reporter (gLUC59). Compared to other available methods, this luciferase‐based strategy requires a shorter processing time, enhances the throughput and improves result analysis of gene‐centered Y1H screens. Here, we described the procedure to perform high‐throughput screens using this novel strategy, which involves a protocol for mating two haploid yeast strains carrying an arrayed TF clone collection and a promoter::gLUC59 reporter, respectively, and a protocol for analyzing gLUC59 activity in the resulting diploid cells. © 2019 by John Wiley & Sons, Inc.

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A yeast one‐hybrid and microfluidics‐based pipeline to map mammalian gene regulatory networks

Abstract: A combined cross-platform approach is presented to experimentally identify and characterize interactions between mouse transcription factors and regulatory elements at unprecedented resolution and throughput.

Cited by 35 publications

References 90 publications

Mitotic chromosome binding predicts transcription factor properties in interphase

Mitotic chromosome binding predicts transcription factor properties in interphase

“Hit‐and‐run”: Transcription factors get caught in the act

High‐Throughput Yeast One‐Hybrid Screens Using a Cell Surface gLUC Reporter

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