Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping

Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Nature Methods (Impact Factor: 32.07). 09/2007; 4(8):659-64. DOI: 10.1038/nmeth1063
Source: PubMed


Yeast one-hybrid (Y1H) assays provide a gene-centered method for the identification of interactions between gene promoters and regulatory transcription factors (TFs). To date, Y1H assays have involved library screens that are relatively expensive and laborious. We present two Y1H strategies that allow immediate prey identification: matrix assays that use an array of 755 individual Caenorhabditis elegans TFs, and smart-pool assays that use TF multiplexing. Both strategies simplify the Y1H pipeline and reduce the cost of protein-DNA interaction identification. We used a Steiner triple system (STS) to create smart pools of 4-25 TFs. Notably, we uniplexed a small number of highly connected TFs to allow efficient assay deconvolution. Both strategies outperform library screens in terms of coverage, confidence and throughput. These versatile strategies can be adapted both to TFs in other systems and, likely, to other biomolecules and assays as well.

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Available from: Vanessa Vermeirssen, Nov 14, 2014
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    • "The concept of GT can date back to World War II, which was first proposed by Dorfman [Dorfman, 1943], for the problem of determining which blood samples contain the syphilis antigen (defective also called positive samples) for numerous soldiers. For now, many biological applications benefit from the GT such as blood testing [Ding-Zhu and Hwang, 2000; Dorfman, 1943; Sobel and Groll, 1959], HIV testing [Hughes- Oliver, 2006; Kim et al., 2007; Westreich et al., 2008], clone library screening [Balding and Torney, 1997; Bruno et al., 1995; Knill et al., 1996], protein–protein interaction mapping [Jin et al., 2007; Jin et al., 2006; Vermeirssen et al., 2007; Xin et al., 2009], drug screening [Jones and Zhigljavsky , 2001; Kainkaryam and Woolf, 2008; Kainkaryam and Woolf, 2009; Wilson-Lingardo et al., 1996] and population genotyping [Erlich et al., 2009a; Erlich et al., 2009b; Patterson and Gabriel, 2009; Prabhu and Pe'Er, 2009]. Combine with GT, fewer DNA libraries are required to detect rare variants. "
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    Genetic Epidemiology 12/2013; 37(8). DOI:10.1002/gepi.21769 · 2.60 Impact Factor
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    • "Mating experiments were performed as described previously (Walhout et al, 2000; Vermeirssen et al, 2007). Briefly, the two 384-well AD-TF plates were transformed into the Ya1867 strain (a kind gift from John Reece-Hoyes and Marian Walhout, University of Massachusetts Medical School, USA). "
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    ABSTRACT: The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.
    Molecular Systems Biology 08/2013; 9(1):682. DOI:10.1038/msb.2013.38 · 10.87 Impact Factor
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    • "We therefore decided to construct a Y1H library consisting exclusively of predicted TF DNAbinding sites from B. cinerea fused to the strong yeast GAL4 TF AD. This is the first time, to our knowledge, that such a strategy has been applied to a filamentous fungus, but similar approaches have been described for the study of TFs in other organisms (Caenorhabditis elegans: Vermeirssen et al., 2007; Arabidopsis thaliana: Mitsuda et al., 2010). We report here the construction of a B. cinerea TF Y1H library, its screening with a botrydial biosynthesis gene promoter and the identification and characterisation of a new TF involved in regulating secondary metabolism gene clusters, carbohydrate metabolism, transport, virulence and detoxification mechanisms. "
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