Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Overview

Journal J Vis Exp

Specialties Biology
General Medicine

Date 2019 Feb 26

PMID 30799854

Citations 2

Authors

Shaleen Shrestha

Xing Liu

Clarissa Stephanie Santoso

Juan Ignacio Fuxman Bass

Affiliations

Soon will be listed here.

Abstract

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a 'DNA-bait' (e.g., promoters, enhancers, silencers, etc.) and a 'TF-prey,' which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against >1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

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References

Brady S, Zhang L, Megraw M, Martinez N, Jiang E, Yi C . A stele-enriched gene regulatory network in the Arabidopsis root. Mol Syst Biol. 2011; 7:459. PMC: 3049412. DOI: 10.1038/msb.2010.114. View

Whitfield T, Wang J, Collins P, Partridge E, Force Aldred S, Trinklein N . Functional analysis of transcription factor binding sites in human promoters. Genome Biol. 2012; 13(9):R50. PMC: 3491394. DOI: 10.1186/gb-2012-13-9-r50. View

Jolma A, Yan J, Whitington T, Toivonen J, Nitta K, Rastas P . DNA-binding specificities of human transcription factors. Cell. 2013; 152(1-2):327-39. DOI: 10.1016/j.cell.2012.12.009. View

Burdo B, Gray J, Goetting-Minesky M, Wittler B, Hunt M, Li T . The Maize TFome--development of a transcription factor open reading frame collection for functional genomics. Plant J. 2014; 80(2):356-66. PMC: 4283594. DOI: 10.1111/tpj.12623. View

Pruneda-Paz J, Breton G, Nagel D, Kang S, Bonaldi K, Doherty C . A genome-scale resource for the functional characterization of Arabidopsis transcription factors. Cell Rep. 2014; 8(2):622-32. PMC: 4125603. DOI: 10.1016/j.celrep.2014.06.033. View

Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T . Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007; 4(8):651-7. DOI: 10.1038/nmeth1068. View

Lambert S, Jolma A, Campitelli L, Das P, Yin Y, Albu M . The Human Transcription Factors. Cell. 2018; 172(4):650-665. DOI: 10.1016/j.cell.2018.01.029. View

Li J, Herskowitz I . Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science. 1993; 262(5141):1870-4. DOI: 10.1126/science.8266075. View

Fuxman Bass J, Pons C, Kozlowski L, Reece-Hoyes J, Shrestha S, Holdorf A . A gene-centered C. elegans protein-DNA interaction network provides a framework for functional predictions. Mol Syst Biol. 2016; 12(10):884. PMC: 5081483. DOI: 10.15252/msb.20167131. View

10.

Carrasco Pro S, Imedio A, Santoso C, Gan K, Sewell J, Martinez M . Global landscape of mouse and human cytokine transcriptional regulation. Nucleic Acids Res. 2018; 46(18):9321-9337. PMC: 6182173. DOI: 10.1093/nar/gky787. View

11.

Sewell J, Fuxman Bass J . Options and Considerations When Using a Yeast One-Hybrid System. Methods Mol Biol. 2018; 1794:119-130. DOI: 10.1007/978-1-4939-7871-7_8. View

12.

Reece-Hoyes J, Barutcu A, McCord R, Jeong J, Jiang L, MacWilliams A . Yeast one-hybrid assays for gene-centered human gene regulatory network mapping. Nat Methods. 2011; 8(12):1050-2. PMC: 3263363. DOI: 10.1038/nmeth.1764. View

13.

Vaquerizas J, Kummerfeld S, Teichmann S, Luscombe N . A census of human transcription factors: function, expression and evolution. Nat Rev Genet. 2009; 10(4):252-63. DOI: 10.1038/nrg2538. View

14.

Fuxman Bass J, Reece-Hoyes J, Walhout A . Gene-Centered Yeast One-Hybrid Assays. Cold Spring Harb Protoc. 2016; 2016(12). PMC: 5443116. DOI: 10.1101/pdb.top077669. View

15.

Sahni N, Yi S, Taipale M, Fuxman Bass J, Coulombe-Huntington J, Yang F . Widespread macromolecular interaction perturbations in human genetic disorders. Cell. 2015; 161(3):647-660. PMC: 4441215. DOI: 10.1016/j.cell.2015.04.013. View

16.

Hens K, Feuz J, Isakova A, Iagovitina A, Massouras A, Bryois J . Automated protein-DNA interaction screening of Drosophila regulatory elements. Nat Methods. 2011; 8(12):1065-70. PMC: 3929264. DOI: 10.1038/nmeth.1763. View

17.

Reece-Hoyes J, Pons C, Diallo A, Mori A, Shrestha S, Kadreppa S . Extensive rewiring and complex evolutionary dynamics in a C. elegans multiparameter transcription factor network. Mol Cell. 2013; 51(1):116-27. PMC: 3794439. DOI: 10.1016/j.molcel.2013.05.018. View

18.

Deplancke B, Mukhopadhyay A, Ao W, Elewa A, Grove C, Martinez N . A gene-centered C. elegans protein-DNA interaction network. Cell. 2006; 125(6):1193-205. DOI: 10.1016/j.cell.2006.04.038. View

19.

Bulyk M, Gentalen E, Lockhart D, Church G . Quantifying DNA-protein interactions by double-stranded DNA arrays. Nat Biotechnol. 1999; 17(6):573-7. DOI: 10.1038/9878. View

20.

Fuxman Bass J, Tamburino A, Mori A, Beittel N, Weirauch M, Reece-Hoyes J . Transcription factor binding to Caenorhabditis elegans first introns reveals lack of redundancy with gene promoters. Nucleic Acids Res. 2013; 42(1):153-62. PMC: 3874175. DOI: 10.1093/nar/gkt858. View