Article

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

ABSTRACT

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.

Download full-text

Full-text

Available from: Vanessa Vermeirssen, Nov 14, 2014
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Aug 2013 · Molecular Systems Biology
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Botrytis cinerea, the grey mould fungus, secretes non-host-specific phytotoxins that kill the cells of many plant species. Phytotoxic assays performed about ten years ago, have highlighted the role in the infection mechanism of one of these secondary metabolites, the sesquiterpene botrydial. We recently showed that BcBOT1 to BcBOT5 genes, which are required for botrydial biosynthesis, are organised into a physical cluster. However, this cluster includes no gene encoding a transcription factor (TF) that might specifically coregulate the expression of BcBOT genes. To identify which TF(s) are implicated in the regulation of this cluster and thereby to decipher DNA-protein interactions in the phytopathogenic fungus B. cinerea, we developed a strategy based on the yeast one-hybrid (Y1H) method. In this study, a Y1H library was generated with the TFs predicted from complete genome sequencing. The screening of this library revealed an interaction between a promoter of the botrydial biosynthesis gene cluster and a new Cys(2)His(2) zinc finger TF, that we called BcYOH1. Inactivation of the BcYOH1 gene and expression analyses demonstrated the involvement of this TF in regulating expression of the botrydial biosynthesis gene cluster. Furthermore, whole-transcriptome analysis suggested that BcYOH1 might act as a global transcriptional regulator of phytotoxin and other secondary metabolism gene clusters, and of genes involved in carbohydrate metabolism, transport, virulence and detoxification mechanisms.
    Full-text · Article · Feb 2013 · Fungal Genetics and Biology
  • Source
    • "CEH-14 and CEH-63 both bind directly and independently to the mbr-1 promoter. When the mbr-1 promoter was tested against the transcription factor array (Reece-Hoyes et al., submitted for publication; Vermeirssen et al., 2007) in a Y1H screen, two of the 30 Fig. 4. DVC axonal defects in the ceh-63(tm541) deletion mutant background. The morphology of DVC was observed from ceh-63 prom ::gfp fusion gene expression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Homeobox proteins are critical regulators of developmental gene transcription and cell specification. Many insights into transcriptional regulation have been gained from studies in the nematode Caenorhabditis elegans. We investigated the expression and regulation of the C. elegans homeobox gene ceh-63, which encodes a single-homeodomain transcription factor of 152 amino acids. ceh-63 is expressed in the interneuron DVC in both sexes, from late embryogenesis through adulthood, and two pairs of uterine cells in reproductive hermaphrodites only. A reporter gene fusion, encoding GFP fused to the full-length CEH-63, also drove weak inconsistent expression in additional unidentified cells in the head and tail. A potential ceh-63 null mutant had no obvious abnormalities, except for a possible increase in subtle defects of the DVC axon projection. No behavioural responses were observed upon either laser ablation of DVC or activation of DVC through light stimulation of channelrhodopsin-2 specifically expressed in this neuron. The function of DVC therefore remains enigmatic. A transcriptional regulatory cascade operating in DVC was defined from the LIM-homeodomain protein CEH-14 through CEH-63 to the helix-turn-helix transcription factor MBR-1. Both CEH-14 and CEH-63 individually bound the mbr-1 promoter in a yeast one-hybrid assay. A model is proposed suggesting that CEH-14 activates ceh-63 and then along with CEH-63 co-ordinately activates mbr-1.
    Full-text · Article · Dec 2011 · Gene
Show more