Yeast one-hybrid assays for gene-centered human gene regulatory network mapping

Abstract

Gateway-compatible yeast one-hybrid (Y1H) assays provide a convenient gene-centered (DNA-to-protein) approach to identify the repertoire of transcription factors that can bind a DNA sequence of interest. We present a set of Y1H resources, including clones for 988 of 1,434 (69%) predicted human transcription factors, for the interrogation of interactions using either low or high-throughput settings. These approaches detect both known and novel interactions between human DNA regions and transcription factors.

A Genome-Scale Resource for the Functional Characterization of Arabidopsis Transcription Factors

“…Tremendous progress has been made in defining the DNA-binding protein landscape using tools such as TF-centered HT-Y1H (Deplancke et al, 2004; Hens et al, 2011; Reece-Hoyes et al, 2011). By taking advantage of the TF collection compatibility with recombination-based cloning (Gateway, Life technologies), we transferred each TF into a Y1H compatible destination plasmid (pDEST22) that carries the GAL4-Activation Domain (GAL4_AD) located 5′ to the TF insertion site.…”

“…While TF-centered approaches such as ChIP-seq can reveal the extent to which a particular TF is involved in genome-wide regulation, other techniques such as high-throughput yeast one-hybrid (HT-Y1H) are promoter-focused and can provide a survey of potential interactors for a single promoter. Reagents for the latter have been developed and applied successfully to study gene regulation in humans, flies ( Drosophila melanogaster ) and worms ( Caenorhabditis elegans ), and are starting to reveal the complexity of combinatorial gene regulation (Deplancke et al, 2004; Hens et al, 2011; Reece-Hoyes et al, 2011). Recently, we implemented this genomic strategy in plants ( Arabidopsis thaliana , Arabidopsis) and identified novel transcriptional components of the circadian clock (Pruneda-Paz et al, 2009) as well as other plant physiological processes (Ito et al, 2012; Li et al, 2012; Niwa et al, 2013).…”

Establishment of Expression in the SHORTROOT-SCARECROW Transcriptional Cascade through Opposing Activities of Both Activators and Repressors

“…Enhanced yeast-one-hybrid (eY1H) assays are an experimental alternative to predictive models, and have been utilized to identify TF-DNA interactions in multiple systems (Gaudinier et al, 2011; Reece-Hoyes et al, 2011a, 2011b). A significant overlap in eY1H interactions and TF-centric chromatin immunoprecipitation (ChIP) peaks has been shown (Fuxman Bass et al, 2015; Reece-Hoyes et al, 2013).…”

Gene-Centered Yeast One-Hybrid Assays

“…The Y1H system can be used to identify protein–DNA interactions (PDIs) with short cis -regulatory elements (as single copy or tandem repeats) (Li and Herskowitz 1993; Deplancke et al 2006; Reece-Hoyes et al 2009) as well as with longer and more complex DNA fragments such as promoters or enhancers (Martinez et al 2008; Arda et al 2010; Reece-Hoyes et al 2011a; Fuxman Bass et al 2014, 2015e). Cloning of the DNA baits to generate reporter constructs can be achieved by traditional restriction enzyme cloning or by recombination-based technologies such as Gateway cloning (Walhout et al 2000b).…”

“…These TF clones can be combined and screened as a “TF minilibrary” (Deplancke et al 2004), but a more efficient technique is to screen these clones individually as an array to ensure that every TF is interrogated and to remove the need to sequence the clones from the positive yeast because the TF identity at each array position is known (Vermeirssen et al 2007). Recently, high-throughput Y1H platforms have been developed for four widely studied organisms ( Caenorhabditis elegans , Drosophila melanogaster , Arabidopsis thaliana , and human) (Gaudinier et al 2011; Hens et al 2011; Reece-Hoyes et al 2011a,b). These platforms use robotics to manipulate AD–TF arrays in a 1536-colony format.…”